Citronellyl butyrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Citronellyl butyrate

EC Number:

205-463-4

EC Name:

Citronellyl butyrate

Cas Number:

141-16-2

Molecular formula:

C14H26O2

IUPAC Name:

citronellyl butyrate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

CBA/N

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 9 weeks for Set 1; 12 weeks for Set 2
- Weight at study initiation: 18.2 to 21.8 g for Set 1; 19.1 - 25.7 g for Set 2
- Housing: 2 to 3 animals per cage in polysulfone cages (200W x 320D x 140H mm)
- Diet: pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C), ad libitum
- Water: tap water (filtered and irradiated by ultraviolet light), ad libitum
- Acclimation period: 4 to 5 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 23.3 in Set 1; 21.1 - 24.7 in Set 2
- Humidity (%): 44.5 - 57.0 in Set 1; 44.9 - 65.0 in Set 2
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: To: Set 1: 9-21 Jun 2016; Set. 2: 4 Oct - 21 Dec 2016

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Range-finding study: 5, 10, 25, 50% (w/v) and 100%
Set 1: 25, 50, 100% (w/v)
Set 2: 1, 5 and 10% (w/v)

No. of animals per dose:

2 (range-finding study), 5 (main study)

Details on study design:

RANGE-FINDING STUDY: Dose selection was based on the consecutive doses and dose levels are selected from a series of appropriate concentrations such as 100, 50, 25, 10 and 5%.
- Compound solubility: The test substance was dissolved in aceton/olive oil in a preliminary solubility test. Therefore, aceton/olive oil was utilized as vehicle for this study.
Two animals were observed per dose group. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to dosing (Day 1) and on the day of necropsy, Day 6. Both ears of each mouse were observed for erythema and scored using erythema score. Ear thickness measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 and Day 6. Additionally, on Day 6, ear weight was determined by balance (TE214S, BS224S, Sartorius, Germany).
- Irritation: not specified
- Systemic toxicity: toxicity was not observed in the high dose group (100%)
- Ear thickness measurements: the ear weight did not show toxicity, but it was increased when compared to the negative control group
- Erythema scores: not specified

MAIN STUDY: As a result of the dose range finding study, toxicity was not observed in the high dosed (100%) animals, thus dose levels of 100, 50, 25% were selected as the high, mid and low doses in the main study.
In addition, the positive and negative control groups were included in the main study.
The doses of the test substance were added at 1, 5, 10% (Main Study, Set 2) after discussion with the sponsor.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ELISA BrdU
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the BrdU labelling index of each test group divided by the BrdU labelling index of the vehicle control group. SI < 1.6 is considered as negative result. SI >= 1.6 is considered as positive result.

The EC1.6 value was used to classify the test substance according to ECETOC Potency classification as follows.
EC1.6 Value(%) ≥ 10 to ≤ 100 -> Weak
EC1.6 Value(%) ≥ 1 to ≤ 10 -> Moderate
EC1.6 Value(%) ≥ 0.1 to ≤ 1 -> Strong
EC1.6 Value(%) -> < 0.1 Extreme
Cindy A et al. Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutaneous and Ocular Toxicology, 2007, 26: 135-145.

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. Two days after the third application on Day 5, an intraperitoneal injection of 0.5 mL (5 mg/mouse) of BrdU solution (10 mg/mL) was made. Approximately 24 h after BrdU injection, the mice were sacrificed and draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal. A single cell suspension was prepared by separation through a nylon mesh. In each case, the target volume of the cell suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1 - 0.2 in the negative control group. BrdU was measured by ELISA using a commercial kit. Briefly, 100 µL of the lymph node cell suspension was added to the wells of a microplate in triplicate. After fixation and denaturation of the cell suspension, anti-BrdU antibody was added to each well. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was added. Absorbance was measured at 370 nm with a reference wavelength of 492 nm.

Negative control animals were dosed with the vehicle, AOO solution.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index.
Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group. Since it was not significant, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.
Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.

Results and discussion

Positive control results:

Body weights: Set 1 and 2: There were no significant differences when compared to the negative control group.
Irritation: Set 1 and 2: The mean erythema score was 0.0–1.9. There were significant increases when compared to the negative control group (p<0.01: Days 3, 4, 5 and 6).
Ear thickness: Set 1: The mean ear thickness was 0.19–0.22 mm. There were significant increases when compared to the negative control group (p<0.01: Day 6). Set 2: The mean value of ear thickness ranged within 0.20–0.21 mm. There was a significant increase when compared to the negative control group (p<0.01: Days 1, 3 and 6).
Ear Weights: Set 1: The mean ear weight was 14.2 mg. There was a significant increase when compared to the negative control group (p<0.01). Set 2: the mean value of ear tissue was 13.9 mg. There was a significant increase when compared to the negative control group (p<0.01).
Stimulation Index: Set 1: The mean stimulation index was 3.10. There was a significant increase when compared to the negative control group (p<0.05). Set 2: The mean value of stimulation index was 4.32. There was a significant increase when compared to the negative control group (p<0.05).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.22

Test group / Remarks:

1% (w/v)

Results 2

Parameter:

SI

Value:

1.1

Test group / Remarks:

5% (w/v)

Results 3

Parameter:

SI

Value:

1.4

Test group / Remarks:

10% (v/w)

Results 4

Parameter:

SI

Value:

1.78

Test group / Remarks:

25% (w/v)

Results 5

Parameter:

SI

Value:

2.15

Test group / Remarks:

50% (w/v)

Results 6

Parameter:

SI

Value:

1.7

Test group / Remarks:

100% (w/v)

Results 7

Parameter:

other: EC1.6

Value:

26.4

Cellular proliferation data / Observations:

IRRITATION, EAR THICKNESS and EAR WEIGHTS:
Erythema Score: Set 1: In the negative control group, the mean value of erythema score was 0.0 prior to dosing until Day 6 after dosing.
In the test substance groups at 25, 50 and 100%, the mean erythema score was 0.0–0.7, 0.0–0.9 and 0.0–1.2, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 4 (25%), p<0.01: Days 4 (50 and 100%), 5 and 6 (25, 50 and 100%)).
Set 2:
In the negative control group, the mean value of erythema score was 0.0 prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of erythema was 0.0, 0.0–1.1 and 0.0–1.0, respectively. There were significant increases when compared to the negative control group (p<0.01: Days 5 and 6 (5 and 10%)).

Ear Thickness: Set 1: In the negative control group, the mean values of ear thickness was 0.19–0.20 mm prior to dosing until Day 6 after dosing.
In the test substance groups at 25, 50 and 100%, the mean ear thickness was 0.19–0.21, 0.20–0.21 and 0.19–0.21 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 6 (25%).
Set 2:
In the negative control group, the mean values of ear thickness was 0.19–0.19 mm prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of ear thickness ranged within 0.20–0.19, 0.20–0.20, 0.20–0.20 mm, respectively. There was a significant increase when compared to the negative control group (p<0.05: Day 1 (1%), Day 3 (5%), p<0.01: Days 1 (5 and 10%), 3 (10%) and 6 (5 and 10%).

Ear weights: Set 1: In the negative control group, the mean ear weight was 12.6 mg.
In the test substance groups at 25, 50 and 100%, the mean ear weight was 13.5, 13.5 and 13.7 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 100%).
Set 2: In the negative control group, the mean value of ear tissue was 12.1 mg.
In the test substance groups at 1, 5, 10%, the mean values of ear tissue were 12.9, 13.3 and 12.6 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 5%).

DETAILS ON STIMULATION INDEX CALCULATION: Set 1: In the negative control group, the mean stimulation index was 1.00.
In the test substance groups at 25, 50 and 100%, the mean stimulation index ± standard deviation was 1.78 ± 0.27, 2.15 ± 0.70 and 1.70 ± 0.38, respectively. There were significant increases when compared to the negative control group (p<0.05: 25, 50 and 100%).
Set 2: In the negative control group, the mean value of stimulation index was 1.00.
In the test substance groups at 1, 5, 10%, the mean values ± standard deviation of stimulation index were 1.22 ± 0.14, 1.10 ± 0.12 and 1.40 ± 0.63, respectively. There were no significant differences when compared to the negative control group.


EC1.6 CALCULATION: EC1.6 =c+[(1.6-d)/(b-d)] x (a-c)
a= The dose concentration with higher SI; b = The higher SI value, c = The dose concentration with lower SI; d = the lower SI value
EC1.6 = 26.4%

CLINICAL OBSERVATIONS: Set 1 and Set 2: There were no abnormal clinical sighs or deaths in any dosing group during the observation period.

BODY WEIGHTS: Set 1: In the negative control group, the mean body weights were 19.3–20.2 g from Day 1 to Day 6 after dosing.
In the test substance groups at 25, 50 and 100%, the mean body weights were 19.1–19.5, 19.5–19.6 and 19.3–19.3 g, respectively. There were no significant differences when compared to the negative control group.
Set 2: In the negative control group, the mean value of body weight ranged within 21.1–21.3 g prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of body weights ranged within 21.7–21.4, 21.6–20.6 and 21.9–20.8 g, respectively. There were no significant differences when compared to the negative control group.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sens Cat 1 required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the local lymph node assay, the test substance revealed a SI >= 1.6 at concentrations of 25, 50 and 100%. The calculated EC1.6 value was 26.4%. Therefore, the test substance is considered as weak sensitiser.

Executive summary:

After treatment of CBA/N mice with the test substance, the test substance produced stimulation indices of 1.22, 1.10, 1.40, 1.78, 2.15 and 1.7 at the concentrations of 1, 5, 10, 25, 50 and 100%, respectively

and it was considered to be a sensitizer (defined as producing a positive response). The EC1.6 value was 26.4%.